Skeletal muscle differentiation and maturation is a complex process involving the synergy of different growth factors and hormones interacting over a broad time period [1-11]. The differentiation process is further complicated by neuronal innervation, where neuron to muscle cell signaling can regulate myosin heavy chain (MHC) gene expression and acetylcholine receptor clustering [12-18]. Consequently, understanding of the role of the growth factors, hormones and cellular interactions in skeletal muscle differentiation would be a key step in generating physiologically relevant tissue engineering constructs, developing advanced strategies for regenerative medicine and integrating functional skeletal muscle with bio-hybrid MEMS devices for non-invasive interrogation in high-throughput screening technologies.
In order for skeletal muscle myotubes developed in vitro to be useful in tissue engineering applications, they must exhibit as many of the functional characteristics of in vivo skeletal muscle fibers as possible. During muscle fiber development in vivo, several critical structural changes occur that indicate functional maturation of the extrafusal myotubes. These changes include sarcomere organization, clustering and colocalization of ryanodine (RyR) and dihydropyridine (DHPR) receptors and MHC class switching [19-23]. Each of these structural changes reflects the physiological maturation of the skeletal muscle and is critical for consistent muscular contraction. For example, organization of the contractile proteins myosin and actin into sarcomeric units gives skeletal muscle myotubes organized and structured contraction, a property lacking in smooth muscle. The organization of sarcomeres in skeletal muscle gives rise to anisotropic and isotropic bands of proteins (A and I bands) and gives skeletal muscle a striated appearance. The clustering and colocalization of RyR and DHPR is indicative of transverse tubule (T-tubule) biogenesis and excitation contraction coupling. This developmental step structurally links electrical excitation to the internal contractile system by providing close apposition of DHPR located in the T-tubule and RyR located in the sarcoplasmic reticulum. Finally, a properly functioning skeletal muscle must express the appropriate MHC proteins required for the task it must perform. For example, different muscle fibers express different MHC proteins depending on the rate of contraction and force generation required by the work to be done. Consequently, skeletal muscle fibers change their MHC expression profiles to best meet the requirements of the body as it matures. Without these modifications, an in vitro model of skeletal muscle maturation cannot achieve full physiological relevance.
One approach for identifying the role of specific growth factors and hormones in muscle differentiation is to develop an in vitro model system consisting of a serum-free medium supplemented with the factors of interest. Such a model provides the opportunity to evaluate the role of each factor individually or in combination with others known or believed to be important in skeletal muscle development. For example, the concentration and/or temporal application of medium components in order to influence the maturation of extrafusal fiber or intrafusal fiber subtypes could be easily investigated.
Employing a non-biological growth substrate such as trimethoxy-silylpropyl-diethylenetriamine (DETA) provides an additional measure of control. DETA is a silane molecule that forms a covalently bonded monolayer on glass coverslips, resulting in a uniform, non-hydrophilic surface for cell growth. The use of DETA surfaces is advantageous from a tissue engineering perspective because it can be covalently linked to virtually any hydroxolated surface, it is amenable to patterning using standard photolithography and it promotes long-term cell survival because it is non-digestible by matrix metalloproteinases secreted by the cells [24, 25].
Previously, studies have demonstrated the usefulness of the DETA silane substrate for in vitro culture systems. Interesting features of the DETA silane are that its molecular geometry does not allow for an ordered nanolayer and may partially mimic the three dimensional features of an extracellular matrix, which may be responsible for robust growth of different cell types on this synthetic substrate [24-31]. Additionally, DETA's non-biological nature supports the analysis of ECM proteins secreted by the cell in response to different in vitro conditions.
We earlier developed a defined system that promoted differentiation of different skeletal muscle phenotypes and resulted in the formation of contractile myotubes. This resulted in short-term survival of the myotubes [25, 28]. We also have developed a novel bio-hybrid technology to integrate functional myotubes with cantilever based bio-MEMS devices for the study of muscle physiology, neuromuscular junction formation and bio-robotics applications for use in a model of the stretch reflex arc [32]. More recently, using our defined model system, we have achieved a significant breakthrough by creating mechanosensitive intrafusal myotubes in vitro [33]. The intrafusal fibers differentiated upon addition of neuregulin 1-β-1 to serum-free medium in our defined system. Intrafusal fibers are the myotubes present in the muscle spindle which functions as the sensory receptor of the stretch reflex circuit [16] and combined with extrafusile fibers represent the primary component necessary to reproduce functional muscle function in vitro.
This system has been utilized as a model for different developmental and functional applications, however, further improvements are necessary to enhance the physiological relevance of the skeletal muscle myotubes [32, 33]. Specifically, in order to create a working model of the stretch reflex arc, myotubes are needed that more accurately represent extrafusal fibers in vivo. A more advanced developmental system for skeletal muscle would have applications in basic science research and tissue engineering. In this study, we have demonstrated sarcomere assembly, the development of the excitation-contraction coupling apparatus and myosin heavy chain (MHC) class switching.
The results disclosed herein suggest we have discovered a group of biomolecules that act together as a molecular switch promoting the transition from embryonic to neonatal MHC expression as well as other structural adaptations resulting in the maturation of skeletal muscle in vitro. The discovery of these biomolecular switches will be a powerful tool in regenerative medicine and tissue engineering applications such as skeletal muscle tissue grafts. It should also be useful in higher content high-throughput screening technology.